Chronic infections and cancer cause CD8 T cell exhaustion, a differentiation state characterized by the inability of CD8 T cells to provide immunological protection. Improving the function of exhausted T cells (TEX) by antagonizing checkpoint inhibitors is a potent immunotherapeutic strategy. However, most patients do not exhibit long-term protection, creating a need for improved immunotherapeutic strategies. Resistance to immunotherapy is mainly attributed to the inability of checkpoint inhibitors to reverse the TEX epigenome. Therefore, epigenetically re-directing TEX differentiation to induce durable responses is key to overcoming resistance to immunotherapy. To date, modifying a single transcriptional pathway has had limited success in providing full TEX reinvigoration and inducing long-term persisting immune responses. Instead, microRNAs (miRs) can have a profound impact on the epigenetic regulation of immune cells; however, the role of miRs in TEX differentiation is understudied. We recently identified miR-29a as a unique memory-associated miR that is repressed in TEX. We demonstrated that ectopic expression of miR-29a enhances TEX persistence, attenuates their exhaustion, and promotes differentiation of a TEX subset with progenitor, stem-like characteristics that responds to immunotherapy. Importantly, our preliminary data suggest that miR-29a alters the epigenetic profile of TEX and promotes durable CD8 T cell responses, in striking contrast to anti-PD-L1 therapy. Mechanistically, we demonstrate that miR-29a directly targets the key DNA demethylating enzyme TET2 and that TET2 overexpression abrogates key effects of miR-29a in TEX differentiation. However, the molecular circuits involving miR-29a and TET2 and the implication of miR-29a-TET2 axis in checkpoint inhibitor therapy is unknown. The goal of this proposal is to uncover the role of miR-29a in targeting the key epigenetic modulator TET2 and, thus, define the role of miR-29a in synergizing with checkpoint inhibitors. Our central hypothesis is that miR-29a regulates DNA methylation by directly targeting TET2, therefore, synergizes with checkpoint inhibitors to re-direct TEX differentiation. Aim 1. Identify the role of the miR-29a-TET2 axis in regulating TEX differentiation. We hypothesize that miR- 29a regulates DNA demethylation, by directly targeting TET2, thereby fundamentally alters TEX differentiation. We will elucidate the TET2-dependent versus TET2-independent effects of miR-29a in TEX differentiation and the downstream effects in DNA methylation. Aim 2. Identify the pathways by which miR-29a synergizes with anti-PD-L1. We hypothesize that miR-29a epigenetically alters the differentiation of the progenitor TEX subset and, thereby, synergizes with anti-PD-L1 to promote durable TEX reinvigoration. We will elucidate the molecular mechanisms of synergy between miR-29a and anti-PD-L1 and we will define the TET2-dependent and TET2-independent roles of miR-29a in synergizing with ant...